Sequence
and Temperature Influence on Kinetics of
DNA Strand Displacement at Gold Electrode Surfaces
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Abstract
Understanding complex contributions
of surface environment to tethered
nucleic acid sensing experiments has proven challenging, yet it is
important because it is essential for interpretation and calibration
of indispensable methods, such as microarrays. We investigate the
effects of DNA sequence and solution temperature gradients on the
kinetics of strand displacement at heated gold wire electrodes, and
at gold disc electrodes in a heated solution. Addition of a terminal
double mismatch (toehold) provides a reduction in strand displacement
energy barriers sufficient to probe the secondary mechanisms involved
in the hybridization process. In four different DNA capture probe
sequences (relevant for the identification of genetically modified
maize MON810), all but one revealed a high activation energy up to
200 kJ/mol during hybridization, that we attribute to displacement
of protective strands by capture probes. Protective strands contain
4 to 5 mismatches to ease their displacement by the surface-confined
probes at the gold electrodes. A low activation energy (30 kJ/mol)
was observed for the sequence whose protective strand contained a
toehold and one central mismatch, its kinetic curves displayed significantly
different shapes, and we observed a reduced maximum signal intensity
as compared to other sequences. These findings point to potential
sequence-related contributions to oligonucleotide diffusion influencing
kinetics. Additionally, for all sequences studied with heated wire
electrodes, we observed a 23 K lower optimal hybridization temperature
in comparison with disc electrodes in heated solution, and greatly
reduced voltammetric signals after taking into account electrode surface
area. We propose that thermodiffusion due to temperature gradients
may influence both hybridization and strand displacement kinetics
at heated microelectrodes, an explanation supported by computational
fluid dynamics. DNA assays with surface-confined capture probes and
temperature gradients should not neglect potential influences of thermodiffusion
as well as sequence-related effects. Furthermore, studies attempting
to characterize surface-tethered environments should consider thermodiffusion
if temperature gradients are involved